title: Analysis of ESCRT functions in exosome biogenesis, composition and secretion highlights the heterogeneity of extracellular vesicles.
abstract: Exosomes are extracellular vesicles (EVs) secreted upon fusion of endosomal multivesicular bodies (MVBs) with the plasma membrane. The mechanisms involved in their biogenesis have not yet been fully identified although they could be used to modulate exosome formation and therefore are a promising tool in understanding exosome functions. We have performed an RNA interference screen targeting 23 components of the endosomal sorting complex required for transport (ESCRT) machinery and associated proteins in MHC class II (MHC II)-expressing HeLa-CIITA cells. Silencing of HRS, STAM1 or TSG101 reduced the secretion of EV-associated CD63 and MHC II but each gene altered differently the size and/or protein composition of secreted EVs, as quantified by immuno-electron microscopy. By contrast, depletion of VPS4B augmented this secretion while not altering the features of EVs. For several other ESCRT subunits, it was not possible to draw any conclusions about their involvement in exosome biogenesis from the screen. Interestingly, silencing of ALIX increased MHC II exosomal secretion, as a result of an overall increase in intracellular MHC II protein and mRNA levels. In human dendritic cells (DCs), ALIX depletion also increased MHC II in the cells, but not in the released CD63-positive EVs. Such differences could be attributed to a greater heterogeneity in size, and higher MHC II and lower CD63 levels in vesicles recovered from DCs as compared with HeLa-CIITA. The results reveal a role for selected ESCRT components and accessory proteins in exosome secretion and composition by HeLa-CIITA. They also highlight biogenetic differences in vesicles secreted by a tumour cell line and primary DCs.
authors.affiliation: Institut Curie Section Recherche, 26 rue d'Ulm, 75248 Paris cedex 05, France.
authors.forename: Marina
authors.initials: M
authors.lastname: Colombo
authors.forename: Catarina
authors.initials: C
authors.lastname: Moita
authors.forename: Guillaume
authors.initials: G
authors.lastname: van Niel
authors.forename: Joanna
authors.initials: J
authors.lastname: Kowal
authors.forename: James
authors.initials: J
authors.lastname: Vigneron
authors.forename: Philippe
authors.initials: P
authors.lastname: Benaroch
authors.forename: Nicolas
authors.initials: N
authors.lastname: Manel
authors.forename: Luis F
authors.initials: LF
authors.lastname: Moita
authors.forename: Clotilde
authors.initials: C
authors.lastname: Théry
authors.forename: Graça
authors.initials: G
authors.lastname: Raposo
mesh_terms: D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D003713:Dendritic Cells / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism*; D055354:Exosomes / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D000949:Histocompatibility Antigens Class II / Q000378:metabolism; D006801:Humans; D016253:Microscopy, Immunoelectron; D057146:Multivesicular Bodies / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D060149:Tetraspanin 30 / Q000378:metabolism
chemical_list: C554586:CD63 protein, human; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D000949:Histocompatibility Antigens Class II; C117020:PDCD6IP protein, human; D034741:RNA, Small Interfering; D060149:Tetraspanin 30
keywords: ALIX; ESCRT; Exosomes; Extracellular vesicles; MHC class II